The various stages of processing that occur after the completion of the fermentation or bioconversion stage, including separation, purification, and packaging of the product

1. By
Assit Prof
Dr. P. Berciyal Golda
VICAS

2. Downstream processing
🠶Introduction:-
The various stages of processing that occur after co
seperation, purification and packaging of products.
The various stages of processing that occur after the
completion of the fermentation or bioconversion stage, including
separation, purification, and packaging of the product

3. Strategies to recovery and purify bio-products
Fermentor
Solid-liquid separation
Recovery
Purification
Supernatant
Cells
Cell products
Cell disruption or rupture
Cell debris
Crystallization and drying

4. Stages in Downstream Processing
I. Removal of Insolubles
II. Product Isolation
III. Product Purification
IV. Product Polishing
V. Product Packaging
 A few product recovery methods may be considered to
combine two or more stages.
For example, expanded bed adsorption accomplishes removal of
insolubles and product isolation in a single step. Affinity
chromatography often isolates and purifies in a single step.

5. l. Removal of Insolubles
• Separation of cells, cell debris or other particulate matter
• Typical operations to achieve this:
1) Filtration
2) Centrifugation
3) Sedimentation
4) Flocculation a process where a solute comes out of solution in the
form of floc or flakes.
5) Gravity settling

6. a. Filtration :-
🠶This is a kind of mechanical operation that used for seperation of
solids from fluids (liquids or gases )by interposing a medium to
porous membrane through which the fluid can pass but solids in
the fluuds are retained .
🠶The solids particles that deposited on the filter form of layer which
is known as filter cake.
🠶 All the solids particles are feeded and stopped by the cake and the
cake grows at qhich particles are brought to its surface .
🠶All the fluids goes through the cake and fluid medium..

8. Continous Rotary Vaccum Filter :-
🠶It is one of the most commonly used type of filter
fermentation..
🠶The drum is pre coated prior to filtration.
🠶A small agent of coagulating is added to the broth before it is
pumped before it is pumped into the filter.
🠶 The drum rotates under vaccum and a thin layer of cells sticks
to the drum.
🠶The thickness of the layer increases in the section designed
for forming the cakes.

10. Points to be considered while
selecting the filter medium :-
🠶Ability to build the soild.
🠶Minimum resistance to flow the filtrate.
🠶Resistance to chemical attack.
🠶Minimum cost.
🠶Long life.

11. b. Centrifugation :-
🠶 Centrifugation is used to separate particles of 100 – 0.1 micrometer from
liquid by gravitational forces.
🠶 It depends on particles size , density difference between the cells and the
broth and viscosity of broth.
🠶 Use of the centrifugal force for the seperation of mixtures.
🠶 More dense components migrate away from the axis of centrifuge .
🠶 Less dense components migrate towards the axis .
Types of centrifuges used are
🠶 Tubular bowl centrifuge
🠶 Multichamber centrifuge
🠶 Disc bowl centrifuge etc………….

13. c. Sedimentation :-
🠶 It is applicable only for large particles greater than 100
micrometer flocs.
🠶It is slow process and takes ~ 3hrs.
🠶 It is used in process like activated sludge effluent treatment.
🠶It is free settling process depends only on gravity.
🠶 Particles settling is a high particle density suspension
(hindered settling ).
🠶 Mostly applicable in every fermentors industries which are
large scale or small scale……

15. d. Flocculation :-
🠶Process where a solute comes out of solution in the form of
flocs or flakes.
🠶Particles finer than 0.1 micrometer in water remain
continoulsy in motion due to electrostatic charge which cause
them to repel each other.
🠶Once their electrostatic charge in neutralized ( use of
coagulant ) the finer particles start to collide and combine
together.
🠶 These larger and heavier flocs of particels are called flocs.

16. e. Gravity settings
• It is apllicable only for large particles greater than 100
micrometer flocs.
• It is a slow process and takes ~3 hours.
• It is used in process like activated sludge effluent
treatment.
• It’s a free settling process depends only on gravity.
• Particles settling is a high particle density
suspension(hindered settling).

17. II. Product Isolation
• Removal of those components whose properties vary markedly
from that of the desired product.
• Water is the chief impurity
a) Isolation steps are designed to remove it (i.e.dialysis)
b) Reducing the volume
c) Concentrating the product.
d) Liquid –liquid extraction, adsorption, ultrafiltration, and
precipitation are some of the unit operations involved.

18. Liquid -Liquid extraction
• It is a separation process that takes the advantage of the relative
solubilities of solute in immiscible solvents.
• Solute is dissolved more readily and becomes more concentrated in
the solvent in which it has a higher solubility.
• A partial separation occurs when a number of solutes have different
relative solubilities in the two solvents used.
• Solvent should be non toxic, selective, inexpensive and immiscible with
broth and should have a high distribution coefficent for the product.

19. Liquid–liquid extraction (LLE) :-
🠶 Liquid–liquid extraction (LLE), also known as solvent extraction and
partitioning, is a method to separate compounds or metal complexes,
based on their
relative solubilities in two different immiscible liquids, usually water (polar)
and an organic solvent (non-polar).
🠶 There is a net transfer of one or more species from one liquid
into another liquid phase, generally from aqueous to organic.
🠶The transfer is driven by chemical potential, i.e. once the transfer is complete,
the overall system of protons and electrons that make up the solutes and
the solvents are in a more stable configuration (lower free energy). The
solvent that is enriched in solute(s) is called extract.
🠶The feed solution that is depleted in solute(s) is called the raffinate.

21. Adsorption
• is a surface phenomenon
• It is the binding of molecules to the surface and different from
absorption.
• The binding to the surface is weak and reversible.
• Compounds containing chromogenic group are usually strongly
adsorbed on activated carbon.
• Common adsorbent used are activated carbon,silica
gel,alumina becoz they present enormous surface areas per
unit weight.

22. Ultrafiltration
UF is basically a pressure-driven separation process.
The operating pressure is usually between 0.1 and 1 MPa.

23. Precipitation
• Formation of a solid in a solution during a chemical
reaction.
• Solid formed is called the precipitate and the liquid
remaining above the solid is called the supernate.

24. Precipitation
• Salts such as ammonium & sodium sulphate are used for
proteins to precipitate.
• Organic solvents methanol used to precipitate dextrans.
• Chilled ethanol and acetone used for protein precipitation.
• Non ionic polymer such as polyethylene glycol used in
precipitation.

25. lll. Product Purification
• Done to separate those contaminants that resemble the product
very closely in physical and chemical properties.
• Expensive to carry out
• Require sensitive and sophisticated equipment
• Significant fraction of the entire downstream processing
expenditure.
• Examples of operations include affinity, size exclusion,
reversed phase chromatography,crystallization and fractional
precipitation.

26. Product purification :-
Purification by
🠶Gel filtration
🠶Ion exchange
🠶Affinity
🠶Hydrophobic interactions.

27. Chromatography
• Separation of mixtures
• Passing a mixture dissolved in a "mobile phase" through a
stationary phase, which separates the analyte to be
measured from other molecules in the mixture and allows
it to be isolated.

28. Ion Exchange Chromatography
• Used charged stationary phase to
separate charged compounds
• Resin that carries charged
functional groups which interact
with oppositely charged groups
of thecompound to be retained.
• FPLC

29. Affinity chromatography:
🠶This is an elegant method for the purification of proteins from a complex
mixture.
🠶Affinity chromatography is based on an interaction of a protein with an
immobilized ligand.
🠶 The ligand can be a specific antibody, substrate, substrate analogue or an
inhibitor. The immobilized ligand on a solid matrix can be effectively used
to fish out complementary structures.
🠶 The protein bound to the ligand can be eluted by reducing their
interaction.
🠶This can be achieved by changing the pH of the buffer, altering the ionic
strength or by using another free ligand molecule.
🠶 The fresh ligand used has to be removed in the subsequent steps

30. Definition: Ion
• Ion is an atom or molecule which has lost or gained one or more
valence electrons, giving it a positive or negative electrical charge.
• Anions are negatively charged ions, formed when an atom gains
electrons in a reaction. Anions are negatively charged because
there are more electrons associated with them than there are
protons in their nuclei.
• Cations are positively charged ions, formed when an atom loses
electrons in a reaction, forming an 'electron hole'.

31. Affinity chromatography
• Affinity chromatography separates the protein of interest on the
basis of a reversible interaction between it and its antibody
coupled to a chromatography bead (here labeled antigen) .
• With high selectivity, high resolution, and high capacity for the
protein of interest, purification levels in the order of several
thousand-fold are achievable.
• The protein of interest is collected in a purified, concentrated form.
Biological interactions between the antigen and the protein of
interest can result from electrostatic interactions, van der Waals'
forces and/or hydrogen bonding. To elute the protein of interest
from the affinity beads, the interaction can be reversed by changing
the pH or ionic strength.
• The concentrating effect enables large volumes to be processed.
The protein of interest can be purified from high
levels of contaminating substances.
• Making antibodies to the protein of interest is expensive, so affinity
chromatography is the least economical choice for production
chromatography.

32. Size exclusion chromatography
• Gel permeation/filtration
• chromatography (GPC)
• Separates molecules
• according to their size
• Low resolution"polishing"
• Tertiary/Quaternary structure(native)

33. Reversed phase chromatography
Reversed-phase chromatography is an elution procedure
used
in liquid chromatography in which
the mobile phase is significantly
more polar than the stationary phase.

34. Definitions: Polarity
• The dipole-dipole intermolecular forces
between the slightly positively-charged end of one molecule
to the negative end of another or the same molecule.
• Molecular polarity is dependent on the difference in
electronegativity between atoms in a compound and the
asymmetry of the compound's structure.

35. Liquid Chromatography
• Mobile phase is a liquid.
• Carried out either in a column or a plane.
• HPLC
• In the HPLC technique, the sample is forced through a
column that is packed with irregularly or spherically shaped
particles or a porous monolithic layer (stationary phase) by a
liquid (mobile phase) at high pressure.

36. HPLC Configuration

37. Crystallization
• process of formation of solid crystals precipitating from a
solution, melt or more rarely deposited directly from a gas.
• chemical solid-liquid separation technique, in which mass
transfer of a solute from the liquid solution to a pure solid
crystalline phase occurs.

38. Cell disruption :-
🠶 Some product are intracellular including many enzymes and
proteins required to disrupt the cell amd rrlease these products.
E.g :- yeast
🠶 Cell disruption can be achieved by both mechanical and non
mechanical methods.
Mechanical :-
🠶 Sonication and liquid shear homogenization etc…..
Non mechanical methods :-
🠶 Autolysis and osmosis shock etc…….

39. Solvent extraction :-
🠶 Reducing the volume of material to be handled and
concentrating the product.
🠶Removal of those components whose properties vary
markedly from that of desired product …
🠶Water Is the Che if impurity .
🠶Liquid liquid extraction
🠶Adsorption
🠶Precipitation
🠶 Are some of the unit operations involved in them….

40. Adsorption
🠶Adsorption is the adhesion of atoms, ions or molecules from a gas,
liquid or dissolved solid to a surface.
🠶 This process creates a film of the adsorbate on the surface of
the adsorbent. This process differs from absorption, in which
a fluid (the absorbate) is dissolved by or permeates a liquid or solid
(the absorbent), respectively.
🠶 Adsorption is a surface phenomenon, while absorption involves the
whole volume of the material. The term sorption encompasses both
processes, while desorption is the reverse of it.
🠶The separation is based on the interaction of the adsorbate with the
adsorbent. The adsorbent is the surface and adsorbate is the
molecules of interest which are getting adsorbed on the adsorbent.

41. Ultrafiltration
🠶Ultrafiltration (UF) is a pressure-driven barrier to suspended solids,
bacteria, viruses, endotoxins and other pathogens to produce water with
very high purity and low silt density.
🠶 Ultrafiltration (UF) is a variety of membrane filtration in which hydrostatic
pressure forces a liquid against a semi permeable membrane.
🠶The variety of membrane filtration in which forces
like pressure or concentration gradients lead to a separation through
a semipermeable membrane.
🠶 Suspended solids and solutes of high molecular weight are retained in
them called retentate, while water and low molecular weight solutes pass
through the membrane in the permeate (filtrate).
🠶 This separation process is used in industry and research for purifying and
concentrating macromolecular (103 - 106 Da) solutions,
especially protein solutions.

42. Precipitation :-
🠶 Precipitation is the most commonly used technique in industry for the concentration of
macromolecules such as proteins and polysaccharides.
🠶 Further, precipitation technique can also be employed for the removal of certain
unwanted byproducts e.g. nucleic acids, pigments.
🠶 Neutral salts, organic solvents, high molecular weight polymers (ionic or non-ionic),
besides alteration in temperature and pH are used in precipitation.
🠶 In addition to these non-specific protein precipitation reactions (i.e. the nature of the
protein is unimportant), there are some protein specific precipitations e.g., affinity
precipitation, ligand precipitation.
🠶 The most commonly used salt is ammonium sulfate, since it is highly soluble, nontoxic to
proteins and low-priced.
🠶 Ammonium sulfate increases hydrophobic interactions between protein molecules that
result in their precipitation.
🠶 The precipitation of proteins is dependent on several factors such as protein
concentration, pH and temperature.

43. Gel-filtration chromatography:
🠶 This is also referred to as size-exclusion chromatography. In this technique,
the separation of molecules is based on the size, shape and molecular
weight.
🠶 The sponge-like gel beads with pores serve as molecular sieves for
separation of smaller and bigger molecules.
🠶 A solution mixture containing molecules of different sizes (e.g. different
proteins) is applied to the column and eluted.
🠶 The smaller molecules enter the gel beads through their pores and get
trapped.
🠶 On the other hand, the larger molecules cannot pass through the pores
and therefore come out first with the mobile liquid (Fig. 20.7).
🠶 At the industrial scale, gel-filtration is particularly useful to remove salts
and low molecular weight compounds from high molecular weight
products.

45. Ion-exchange chromatography:
🠶 It involves the separation of molecules based on their surface charges.
🠶 Ion-exchangers are of two types (cation- exchangers which have
negatively charged groups like carboxymethyl and sulfonate, and anion-
exchangers with positively charged groups like diethylaminoethyl (DEAE).
🠶 The most commonly used cation-exchangers are Dowex HCR and
Amberlite IR, the anion-exchangers are Dowex SAR and Amberlite IRA.
🠶 In ion-exchange chromatography, the pH of the medium is very crucial,
since the net charge varies with pH.
🠶In other words, the pH determines the effective charge on both the
target molecule and the ion-exchanger.
🠶The ionic bound molecules can be eluted from the matrix by changing
the pH of the eluant or by increasing the concentration of salt solution.
🠶 Ion-exchange chromatography is useful for the purification of antibiotics,
besides the purification of proteins.

46. Product formulations :-
🠶 Formulation broadly refers to the maintenance of activity and stability of a
biotechnological products during storage and distribution.
🠶 The formulation of low molecular weight products (solvents, organic acids) can be
achieved by concentrating them with removal of most of the water.
🠶 For certain small molecules, (antibiotics, citric acid), formulation can be done by
crystallization by adding salts.
🠶 Proteins are highly susceptible for loss of biological activity; hence their formulation
requires special care.
🠶 Certain stabilizing additives are added to prolong the shelf life of protein. The stabilizers
of protein formulation include sugars (sucrose, lactose), salts (sodium chloride,
ammonium sulfate), polymers (polyethylene glycol) and polyhydric alcohols (glycerol).
🠶 Proteins may be formulated in the form of solutions, suspensions or dry powders.

47. lV.Product Polishing
• End with packaging of the product in a form that is
• stable, easily transportable and convenient.
• Crystallization
• Desiccation
• Lyophilization
• Spray drying
• May include:
• Sterilization of the product
• Remove or deactivate trace contaminants which might compromise
product safety viruses or depyrogenation

48. lyophilization
• freezing the material
• reducing the surrounding pressure and adding enough heat to
allow the frozen water in the material to sublime directly from
the solid phase to gas.

49. V. Product packaging :-
🠶Drying
🠶Freezing
🠶Space drying

50. Thanking you ……